This invention relates to vascular damaging agents and particularly to use in the preparation of agents for treatment of neovascularisation of a group of colchinol derivatives some of which are new compounds.
Formation of new vasculature by angiogenesis is a key pathological feature of several diseases (J Folkman, New England Journal of Medicine 333, 1757 (1995)). For example, for a solid tumour to grow it must develop its own blood supply upon which it depends critically for the provision of oxygen and nutrients; if this blood supply is mechanically shut off the tumour undergoes necrotic death. Neovascularisation is also a clinical feature of skin lesions in psoriasis, of the invasive pannus in the joints of rheumatoid arthritis patients and of atherosclerotic plaques. Retinal neovascularisation is pathological in macular degeneration and i diabetic retinopathy. In all these diseases reversal of neovascularisation by damaging the newly-formed vascular endothelium is expected to have a beneficial therapeutic effect.
Colchinol derivatives for example N-acetyl-colchinol are known. Anti-tumour effects have been noted on animal models (see for examplexe2x80x94JNCI (Journal National Cancer Institute) Page 379-392 1952, Vol 13). However, the effect studied was that of gross damage (haemorrhage, softening and necrosis) and there is no suggestion of treatment of inappropriate angiogenesis by destruction of neovasculature.
A search of Chemical Abstracts (post 1955) based on the substructure 
revealed a number of colchinol related structures.
To the extent that any of these compounds have been studied for anti-cancer activity it is because tubulin binding agents might be expected to be anti-mitotic and therefore to have a direct effect on tumour cells.
In the course of the work on the present invention, the issue of the relevance of tubulin-binding properties to possible effectiveness as anti-vascular agent was studied but no predictability was found. Thus docetaxel (Lancet, 344, 1267-1271, 1994), which is a tubulin-binding agent, had no vascular-damaging effects even when administered at its Maximum Tolerated Dose. Even when the present inventors tested some compounds structurally related to the present invention, the therapeutic window (ratio of MTD (Maximum tolerated dose) to MED (Minimum effective dose)) was found to be too small for potential clinical effectiveness.
According to the present invention there is provided the use of colchinol derivatives for the preparation of compositions for the treatment of diseases involving angiogenesis in which the colchinol derivative has the formula 
wherein
R1, R2, R3 and R5 are each independently H. optionally substituted alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, alkanoyl, PO3H2;
X is carbonyl (CO), thiocarbonyl (CS), methylene (CH2) or a group CHR4 
R4 is OH, O-alkyl or NR8R9;
R5 and R7 are each independently H, alkyl, halogen, hydroxy, alkoxy, nitro or amino;
R8 is H, optionally substituted alkyl, cycloalkyl, alkanoyl, thioalkanoyl, aryl, heteroaryl, arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, arylaminocarbonyl, alkylsulphonyl, arylsulphonyl, aminosulphonyl, alkylaminosulphonyl, dialkylaminosulphonyl or arylaminosulphonyl;
and R9 is H, alkyl or cycloalkyl
and the pharmaceutically acceptable salts, solvates, and hydrates thereof.
It is believed, though this is not limiting on the invention, that the use of compounds of the invention damages newly-formed vasculature, for example the vasculature of tumours, thus effectively reversing the process of angiogenesis as compared to known anti-angiogenic agents which tend to be less effective once the vasculature has formed.
Certain of these compounds are novel. In one embodiment the novel compounds are those of formula I in which at least one of R1, R2, R3, R6 is PO3H2. In a particular preferred embodiment R6 is PO3H2. Particularly preferred are compounds defined by the formula 
wherein
R1, R2 and R3 are each independently H, optionally substituted alkyl, cycloalkyl, alkenyl, alkynyl, alkanoyl, or PO3H2;
R6 is PO3H2;
R4 is H or NR8R9;
R5 and R7 are each independently H, alkyl, halogen, alkoxy, nitro or amino;
R8 is H, optionally substituted alkyl, cycloalkyl, alkanoyl, thioalkanoyl, aryl, heteroaryl, arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, arylaminocarbonyl, alkylsulphonyl, arylsulphonyl, aminosulphonyl, alkylaminosulphonyl, dialkylaminosulphonyl or arylaminosulphonyl;
and R9 is H, alkyl or cycloalkyl,
and the pharmaceutically acceptable salts, solvates and hydrates thereof.
In another aspect of the invention the novel compounds are of formula 
wherein
R1, R2 and R3 are each independently H, optionally substituted alkyl, cycloalkyl, alkenyl, alkynyl, alkanoyl or PO3H2;
R6 is H, optionally substituted alkyl, cycloalkyl, alkenyl, akynyl or PO3H2;
R4 is H or NR8R9;
R5 and R7 are each independently H, alkyl halogen, nitro or amino;
R8 is H, optionally substituted alkyl, cycloalkyl, alkanoyl, thioalkyl, aryl, heteroaryl, arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, arylaminocarbonyl, alkylsulphonyl, arylsulphonyl, aminosulphonyl, alkylaminosulphonyl, dialkylaminosulphonyl or arylaminosulphonyl;
and R9 is H, alkyl or cycloalkyl, with the proviso that, when R1, R2 and R3 are all methyl groups and R4 is hydrogen, acetylamino, acetylmethylamino, amino, methylamino or dimethylamino then R6 is not hydrogen, methyl or hydroxyethyl, or acetoxyethyl,
and the pharmaceutically acceptable salts, solvates and hydrates thereof.
Preferred compounds used in the invention and of the invention are those in which R1, R2 and R3 are alkyl and those in which R4 is acylamino.
As used herein the term xe2x80x9calkylxe2x80x9d, (including any aliphatic structure chain related to alkyl) means a straight or branched-chain group containing from one to seven, preferably a maximum of four, carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, t-butyl and pentyl. Optional substituents which may be present on the alkyl groups include one or more substituents selected from halogen, amino, monoalkylamino, dialkylamino, hydroxy, alkoxy, alkylthio, alkylsulphonyl, acylamino, alkoxycarbonylamino, alkanoyl, acyloxy, carboxyl, sulphate or phosphate groups. Examples of alkoxy groups are methoxy, ethoxy, propoxy, isopropoxy, butoxy and t-butoxy. The term xe2x80x9chalogenxe2x80x9d means fluorine, chlorine, bromine or iodine.
An alkenyl group is an olefinic group containing from two to seven carbon atoms for example methylene, ethylene, n-propylene, i-propylene, n-butylene, i-butylene, s-butylene and t-butylene. An alkynyl group is of a group of 2-7 carbon atoms for example ethynyl, propynyl or butynyl group.
The term aryl alone or in combination means an unsubstituted phenyl group or a phenyl group carrying one or more, preferably one to three, substituents examples of which are halogen, alkyl, haloalkyl, hydroxy, nitro, cyano, amino and alkoxy. A haloalkyl group can carry one or more halogen atoms with the examples of such groups being trifluoromethyl and dichloromethyl.
The term heteroaryl is defined herein as a mono- or bi-cyclic aromatic group containing one to four heteroatoms selected in any combination from N, S or O atoms and a maximum of 9 carbon atoms. Examples of heteroaryl groups include pyridyl, pyrimidyl, furyl, thienyl, pyrrolyl, pyrazolyl, indolyl, benzofuryl, benzothienyl, benzothiazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, imidazolyl, triazolyl, quinolyl and isoquinolyl groups.
The term aralkyl is defined herein as an alkyl group, as previously defined, in which one of the hydrogen atoms is replaced by an aryl or heteroaryl group as defined herein.
Where one or more functional groups in compounds of formulae I, II, IIA are sufficiently basic or acidic the formation of salts is possible. Suitable salts include pharmaceutically acceptable salts for example acid addition salts including hydrochlorides, hydrobromides, phosphates, sulphates, hydrogen sulphates, alkylsulphonates, arylsulphonates, acetates, benzoates, citrates, maleates, fumarates, succinates, lactates and tartrates, salts derived from inorganic bases including alkali metal salts such as sodium or potassium salts, alkaline earth metal salts such as magnesium or calcium salts, and salts derived from organic amines such as morpholine, piperidine or dimethylamine salts.
Those skilled in the art will recognise that compounds of formulae I, II, IIA may exist as stereoisomers and/or geometrical isomers and accordingly the present invention includes all such isomers and mixtures thereof.
One useful group of compounds includes those in which R1, R2 and R3 are each alkyl.
Another useful group of compounds includes those in which R1, R2 and R3 are each alkyl and R5 and R7 are each hydrogen. A particularly useful subset of this group includes compounds in which R1, R2 and R3 are each methyl and R6 is hydrogen, alkyl or PO3H2.
Particularly useful compounds according to the invention include:
N-Acetylcolchinol-O-phosphate and its salts, solvates and hydrates.
Compounds of formulae I, II or IIA may be prepared by a number of processes as generally described below and more specifically in the Examples hereinafter. In the following process description the symbols R1, R2, R3, R4, R5, R6 and R7 when used in the formulae depicted are to be understood to represent those groups described above in relation to formulae I, II or IIA unless otherwise indicated. In the schemes described below it may be necessary to employ protecting groups which are then removed during the final stages of the synthesis. The appropriate use of such protecting groups and processes for their removal will be readily apparent to those skilled in the art.
Thus according to a further aspect of the invention compounds of formulae II or IIA in which R5, R6 and R7 are each hydrogen may be prepared by treatment of a compound of formula (2) with alkaline hydrogen peroxide. The reaction may be conveniently performed in aqueous sodium hydroxide solution in the absence or presence of a cosolvent such as an alcohol, for example ethanol, at a temperature in the range for example 0-100xc2x0 C. preferably at or near to 60xc2x0 C. 
Intermediates of formulae (2) may be prepared by acid hydrolysis of compounds of formulae (3). The reaction is conveniently carried out in an aqueous acid such as hydrochloric acid at an elevated temperature, for example at or near 100xc2x0 C. 
Compounds of formula (3) are either known or can be prepared from colchicine by conventional procedures.
Compounds of formulae I, II or IIA may also be prepared from other compounds of formulae I, II or IIA, by chemical modification. Examples of such chemical modifications that may be applied are standard alkylation, arylation. heteroarylation, acylation, thioacylation, sulphonylation, sulphation, phosphorylation, aromatic halogenation and coupling reactions. These reactions may be used to add new substituents or to modify existing substituents. Alternatively, existing substituents in compounds of formulae I, II or IIA may be modified by, for example oxidation, reduction, elimination, hydrolysis or other cleavage reaction to yield other compounds of formulae I, II or IIA.
Thus for example a compound of formulae II or IIA containing an amino group may be acylated on the amino group by treatment with, for example, an acyl halide or anhydride in the presence of a base, for example a tertiary amine base such as triethylamine, in for example, a solvent such as a hydrocarbon solvent e.g. dichloromethane at a temperature in the range for example xe2x88x9230xc2x0 C. to 120xc2x0 C., conveniently at or near ambient temperature.
In another general example of an interconversion process an amino group in a compound of formulae II or IIA may be sulphonylated by treatment with, for example, an alkyl or aryl sulphonyl chloride or an alkyl or aryl sulphonic anhydride in the presence of a base, for example a tertiary amine base such as triethylamine, in for example a solvent such as a hydrocarbon solvent e.g. dichloromethane at a temperature in the range for example xe2x88x9230xc2x0 C. to 120xc2x0 C., conveniently at or near ambient temperature.
In a further general example a compound of formulae II or IIA containing a hydroxy group can be converted into the corresponding dihydrogenphosphate ester by treatment with for example di-tert-butyl diethylphosphoramidite in the presence of a suitable catalyst for example tetrazole. In a solvent such as an ether solvent for example tetrahydrofuran at a temperature in the range xe2x88x9240 to 40xc2x0 C., conveniently at or near room temperature, followed by treatment with an oxidising agent for example 3-chloroperoxy benzoic acid at a temperature in the range xe2x88x9278xc2x0 C. to 40xc2x0 C. preferably xe2x88x9240 to xe2x88x9210xc2x0 C. The resulting intermediate phosphate triester is treated with an acid for example trifluoroacetic acid in a solvent such as a chlorinated solvent e.g. dichloromethane at a temperature in the range xe2x88x9230 to 40xc2x0 C. conveniently at or near 0xc2x0 C. to give the compound of formula (2) containing a dihydrogenphosphate ester.
In a further general example a compound of formula (2) containing an amide can be hydrolysed by treatment with for example an acid such as hydrochloric acid in a solvent such as an alcohol, for example methanol at an elevated temperature conveniently at the reflux temperature.
In another general example an O-alkyl group may be cleaved to the corresponding alcohol (OH) by reaction with boron cribromide in a solvent such as a chlorinated solvent e.g. dichioromethane at a low temperature e.g. around xe2x88x9278xc2x0 C.
In a further general example compounds of formulae II or IIA may be alkylated by reaction with a suitable alkylating agent such as an alkyl halide, an alkyl toluenesulphonate, an alkyl methanesulphonate or an alkyl triflate. The alkylation reaction can be carried out in the presence of a base for example an inorganic base such as a carbonate e.g. caesium or potassium carbonate, a hydride such as solitaii hydride or an alkoxide such as potassium t-butoxide in a suitable solvent such as an aprotic solvent e.g. dimethylformamide or an ether solvent such as tetrahydrofuran at a temperature of around xe2x88x9210 to 80xc2x0 C.
Preparation of a compound of formulae II or IIA as a single enantiomer or, where appropriate, diastereomer may be effected by synthesis from an enantiomerically pure starting material or intermediate or by resolution of the final product in a conventional manner.
Acid addition salts of the compounds of formulae II or IIA are prepared in a conventional manner by treating a solution or suspension of the free base II or IIA with about one equivalent of a pharmaceutically acceptable acid. Salts of compounds of formulae I, II or IIA derived from inorganic or organic bases are prepared in conventional manner by treating a solution or suspension of the free acid I, II or IIA with about one equivalent of a pharmaceutically acceptable organic or inorganic base. Alternatively both acid addition salts and salts derived from bases may be prepared by treatment of the parent compound with the appropriate ion-exchange resin in a standard fashion. Conventional concentration and recrystallisation techniques are employed in isolating the salts.
Compounds according to the invention are able to destroy tumour vasculature and vasculature that has been newly formed while leaving unaffected normal, mature vasculature. The ability of the compounds to act in this way may be determined by the tests described in the Examples hereinafter.
The compounds according to the invention are thus of particular use in the prophylaxis and treatment of cancers involving solid tumours and in the prophylaxis and treatment of diseases where inappropriate angiogenesis occurs such as diabetic retinopathy, psoriasis, rheumatoid arthritis, atherosclerosis and macular degeneration.
The compounds of the invention may be administered as a sole therapy or in combination with other treatments. For the treatment of solid tumours compounds of the invention may be administered in combination with radiotherapy or in combination with other anti-tumour substances for example those selected from mitotic inhibitors, for example vinblastine, paclitaxel and docetaxel; alkylating agents, for example cisplatin, carboplatin and cyclophosphamide, antimetabolites, for example 5-fluorouracil, cytosine arabinoside and hydroxyurea; intercalating agents for example adriamycin and bleomycin; enzymes, for example asparaginase; topoisomerase inhibitors for example etoposide, topotecan and irinotecan; thymidylate synthase inhibitors for example raltitrexed; biological response modifiers for example interferon; antibodies for example edrecolomab, and anti-hormones for example tamoxifen. Such combination treatment may involve simultaneous or sequential application of the individual components of the treatment.
For the prophylaxis and treatment of disease the compounds according to the invention may be administered as pharmaceutical compositions selected with regard to the intended route of administration and standard pharmaceutical practice. Such pharmaceutical compositions may take a form suitable for oral, buccal, nasal, topical, rectal or parenteral administration and may be prepared in a conventional manner using conventional excipients. For example for oral administration the pharmaceutical compositions may take the form of tablets or capsules. For nasal administration or administration by inhalation the compounds may be conveniently delivered as a powder or in solution. Topical administration may be as an ointment or cream and rectal administration may be as a suppository. For parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion) the composition may take the form of, for example, a sterile solution, suspension or emulsion.
The dose of a compound of the invention required for the prophylaxis or treatment of a particular condition will vary depending on the compound chosen, the route of administration, the form and severity of the condition and whether the compound is to be administered alone or in combination with another drug. Thus the precise dose will be determined by the administering physician but in general daily dosages may be in the range 0.001 to 100 mg/kg preferably 0.1 to 50 mg/kg.
The following tests were used to demonstrate the activity and selectivity of compounds according to the invention.
Activity Against Tumour Vasculature Measured by Radioactive Tracer.
The following experiment demonstrates the ability of the compounds to damage selectively tumour vasculature.
Subcutaneous CaNT tumours were initiated by injecting 0.05 ml of a crude tumour cell suspension, approximately 106 cells, under the skin overlying the rear dorsum of 12-16 week-old mice. The animals were selected for treatment after approximately 3-4 weeks, when their tumours reached a geometric mean diameter of 5.5-6.5 mm. Compounds were dissolved in sterile saline and injected intraperitoneally in a volume of 0.1 ml per 10 g body weight. Tumour perfusion was measured 6 hours after intraperitoneal administration in tumour, kidney, liver, skin muscle, gut and brain by the 86RbCI extraction technique (Sapirstein, Amer J Physiol, 193, 161-168, 1958). Tissue radioactivity measured 1 minute after an intravenous injection of 86RbCI was used to calculate relative blood flow as a proportion of cardiac output (Hill and Denekamp, Brit J Radiol, 55, 905-913, 1982). Five animals were used in control and treated groups. Results were expressed as a percentage of the blood flow in the corresponding tissues in vehicle treated animals.
Activity Against Tumour Vasculature Measured by Fluorescent Dye.
The following experiment further demonstrates the ability of the compounds to damage tumour vasculature.
Tumour functional vascular volume in CaNT tumour-bearing ice was measured using the florescent dye Hoechst 33342 according to the method of Smith et al (Brit J Cancer 57, 247-253, 1988). Five animals were used in control and treated groups. The fluorescent dye was dissolved in saline at 6.25 mg/ml and injected intravenously at 10 mg/kg 6 hours after intraperitoneal drug treatment. One minute later, animals were killed and tumours excised and frozen; 10 xcexcm sections were cut at 3 different levels and observed under UV illumination using an Olympus microscope equipped with epifluorescence. Blood vessels were identified by their fluorescent outlines and vascular volume was quantified using a point scoring system based on that described by Chalkley, (J Natl Cancer Inst, 4, 47-53, 1943). All estimates were based on counting a minimum of 100 fields from sections cut at the 3 different levels. Compounds of the invention reduced tumour functional vascular volume by greater than 20% at doses of 50 mg/kg or below.